BION1 is a novel ion channel

ABSTRACT

Polymorphisms are identified in a human gene which encodes a voltage-gated ion channel. The gene maps to the same portion of human chromosome 13q22 to which genes for schizophrenia susceptibility, Bipolar Disorder, and Panic Disorder Syndrome have been mapped. The polymorphisms are used to follow inheritance of the susceptibilities within families. They are also used to identify affected probands. A complete mRNA sequence and genomic structure provide insights into function and relationship to other ion channels.

[0001] This application claims priority from Provisional Application Serial No. 60/300,101 filed Jun. 25, 2001, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention is related to ion channels expressed in brain tissue. In particular it is related to ion channels which are associated with susceptibility to psychiatric disorders.

BACKGROUND OF THE INVENTION

[0003] It has long been known that the flow of ions in and out of cells in the nervous system is crucial for their activity, and that specific ion channels are largely responsible for this movement. Ion channels are generally gated (with the exception of the K⁺ leak channels), either by ligand binding or by voltage. Ligand binding ion channels convert extracellular chemical signals into electrical signals, while voltage gated ion channels, particularly Na⁺ channels, play a key role in action potential propagation. The voltage gated Ca⁺⁺ channel provides the only known means of converting electrical signals into chemical signals. We describe here the isolation and characterization of BION1 (Brain Ion 1), a novel voltage gated channel protein of unknown ion specificity. Various lines of evidence are presented that link the gene encoding this protein with a schizophrenia susceptibility locus on chromosome 13q32.

[0004] Schizophrenia is a serious disorder characterized by severe psychotic symptoms, and is fairly common, affecting ˜1% of the general population. The illness often develops in young adults who were previously normal, and is characterized by a constellation of symptoms including hallucinations and delusions (psychotic symptoms) and symptoms such as severely inappropriate emotional responses, disordered thinking and concentration, erratic behavior, as well as social and occupational deterioration (Andreasen, 1995). Family, twin and adoption studies indicate that schizophrenia is primarily (71%) genetic. The genetic component of schizophrenia is complex, polygenic, and involves epistatic interaction between loci. Mapping studies reveal that this genetic component can be mapped to several distinct genetic loci, including SCZD7 on 13q32 (Blouin et al. 1998, Pulver et al. 1998, Shaw et al. 1998, Brzustowicz et al. 1999, Brzustowicz et al. 2000).

[0005] Recently, a genetic linkage between 13q32 and a second syndrome, Panic Disorder Syndrome (Weissman et al. 2000) has been established. Panic Disorder (anxiety neurosis) is also genetic, with a prevalence of 1-3%, and is symptomatically represented by recurrent episodes of sudden apprehension and associated autonomic symptoms involving the cardio-respiratory system, nervous system, and gastrointestinal system. Panic Disorder Syndrome occurs in a subset of Panic Disorder patients, and is additionally associated with an increased rate of mitral valve prolapse, bladder/kidney problems, serious headaches and/or thyroid problems. When these additional symptoms are scored, a high LOD (likelihood of linkage) score was obtained with one marker, D13S779, which is also at 13q32. Interestingly, when only Panic Disorder was considered, there was no significant LOD with this marker, suggesting that panic disorder is also polygenic (Knowles et al. 1998). While multiple case reports of familial association of schizophrenia and Panic Disorder exist in the literature, statistical association of the two disorders has not been performed. Bipolar Disorder, another serious psychiatric disorder, has also been found to be genetically linked to a locus at or near chromosome 13q32.

[0006] There is a need in the art for additional tools for diagnosing and studying psychiatric disorders, including schizophrenia, Bipolar Disorder, and Panic Disorder Syndrome.

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to provide an isolated and purified polynucleotide which encodes human BION1.

[0008] It is another object of the present invention to provide a method of determining susceptibility to Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome.

[0009] It is an object of the present invention to provide an isolated and purified polynucleotide comprising at least 18 contiguous nucleotides of a human BION1 coding sequence.

[0010] Another object of the invention is to provide an isolated and purified human BION1 protein.

[0011] Another object of the invention is to provide an isolated and purified human BION1 polypeptide comprising at least 6 amino acids of a human BION1 protein.

[0012] Another object of the invention is to provide a method of producing human BION1.

[0013] Another object of the invention is to provide a method of screening test substances for candidates useful in treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome.

[0014] These and other objects of the invention are achieved by providing one or more of the embodiments shown below. In one embodiment of the invention an isolated and purified polynucleotide which encodes human BION1 is provided. The polynucleotide encodes an amino acid sequence as shown in SEQ ID NO:1 or a polymorphic variant found in a schizophrenic, Bipolar Disorder, or Panic Disorder Syndrome patient.

[0015] Another aspect of the invention is an isolated and purified polynucleotide comprising at least 18 contiguous nucleotides of a human BION1 coding sequence. The polynucleotide comprises at least one codon identified in Table 1, which codon is for an amino acid found in human BION1 proteins at the corresponding position.

[0016] Still another embodiment provided by the present invention is an isolated and purified human BION1 protein. The protein comprises an amino acid sequence as shown in SEQ ID NO:1 or a polymorphic variant found in Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome patients.

[0017] According to yet another embodiment of the invention, an isolated and purified human BION1 polypeptide comprising at least 6 amino acids of a human BION1 protein is provided. The polypeptide comprises an amino acid sequence found in humans but not in rats as identified in Table 1.

[0018] According to another embodiment of the invention an isolated and purified polynucleotide of a human BION1 gene is provided. The polynucleotide comprises a polymorphic nucleotide identified in Table 2 or Table 3.

[0019] Even another embodiment provided by the present invention is a method of producing human BION1. A host cell comprising a vector which encodes a human BION1 protein is cultured under conditions for expression of BION1 from the vector. BION1 protein is then collected from the cultured host cells or culture medium.

[0020] According to another aspect of the invention a method is provided for screening test substances for candidates useful in treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome. A human BION1 protein is contacted with a test substance. Test substances are determined which bind to the human BION1 protein. A test substance is identified as a candidate drug useful for treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome if it binds to human BION1.

[0021] Thus the present invention provides diagnostic and drug development tools for addressing psychiatric disorders for which genetic and pharmacologic interventions are needed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1a: cDNA sequence of human BION1. The complete coding sequence is underlined, running from nucleotide 230 to 5443..

[0023]FIG. 1b: Protein sequence of human BION1. Protein sequence of human BION1. The positions of the transmembrane domains and loops are shown underlined. The residues in the loop thought to be responsible for ion selectivity (EEKE) are in red bold face.

[0024]FIG. 2a: The predicted secondary structure of BION1, showing its predicted topology within the cell membrane. Additionally, the predicted pore structure of Bion1, Na_(v), and Ca_(v) channels is shown

[0025]FIG. 2b: Predicted Secondary structure of BION1, showing its predicted topology within the cell membrane. Additionally, the predicted pore structure of Bion1, Na_(v), and Ca_(v) channels is shown.

[0026]FIG. 3a: Amino acid residues thought to be crucial in determining ion selectivity demonstrate the relationship between BION1, Na⁺ channels (type II from brain), and Ca⁺⁺ channels (L-type from heart). Note the conserved DEKA in Na⁺ channels and EEEE in Ca⁺⁺ channels is replaced by the novel combination EEKE.

[0027]FIG. 3b: Phylogenetic tree relating different members of the voltage-gated ion channel superfamily. Alignment of the amino acid sequences of S4 regions of different members of the voltage-gated ion channels was generated using CLUSTALW.

[0028]FIG. 4a: Northern blot of rat poly(A)⁺ RNA isolated from a variety of tissues probed with Bion1. Highest expression is in brain and pancreas (not shown), although significant expression is also seen in heart, kidney, small intestine and placenta.

[0029]FIG. 4b: Northern blot of rat poly(A)⁺ RNA isolated from different parts of the brain probed with Bion1. Densitometry readings relative to thalamic expression are given.

[0030]FIG. 5a: In situ hybridization of BION1 to rat brain sections. Note the strong signal in the hippocampus (hip) in panel A.

[0031]FIG. 5b: Emulsion autoradiogram of in situ hybridization of BION1 to rat brain sections. Note strong signal over the neuronal layer of the hippocampus (panel A).

[0032]FIG. 6a: Fluorescent in situ hybridization with BION1 showing hybridization to human chromosome 13q22. DAPI DNA staining in blue and the FISH signal in red.

[0033]FIG. 6b: The same karyotype as in FIG. 6a has been alu-banded in green.

[0034]FIG. 7: BION1 gene structure. Protein coding sequence is shown in shaded.

[0035]FIG. 8: BION1 polymorphism analysis. The positions of all polymorphisms found are marked with an asterisk. Intronic polymorphisms are indicated by minus signs in the name (e.g., T(−221)C, indicating that it is 221 nt from the intron/exon boundary).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] We have determined the sequence of the human homolog of the rat BION1 gene, a voltage-gated ion channel with novel ion specificity sequence. We have localized this gene to human chromosome 13q32, where a schizophrenia susceptibility gene (SCZD7), Bipolar Disorder locus, and Panic Disorder Syndrome locus map genetically. Moreover, the closest marker to these genetic loci, D13S779, is contained within a YAC clone that also contains BION1. We have shown by Northern blot analysis that rat BION1 RNA is expressed in the brain, consistent with a neuroactive protein. Interestingly, it is also expressed moderately in the heart; the subset of Panic Disorder patients that comprise the Panic Disorder Syndrome exhibit a high incidence of mitral valve prolapse. In situ hybridization to rat brain sections confirmed that staining was chiefly confined to neuronal cell types. We have identified the genomic structure of BION1 for all 42 exons. Sequencing of 13 exons, which comprises 27% of the coding region has revealed 3 exonic, 4 intronic, and 4 5′UTR polymorphisms.

[0037] An isolated and purified polynucleotide according to the present invention typically encodes human BION1, which has an amino acid sequence as shown in SEQ ID NO:1. Polymorphic variants found in a schizophrenic, Bipolar Disorder, or Panic Disorder Syndrome patient are also included within the polynucleotides contemplated. The polynucleotides can be cDNA, genomic DNA, RNA or other forms of polynucleotides. The polynucleotide can be in an expression vector or a non-expression vector, such as a YAC clone, a BAC clone, a P1 clone, and the like. Preferably a genomic clone also contains marker D135779, which has been found to be closely linked to Panic Disorder Syndrome and schizophrenia. Preferably the polynucleotide includes at least 12, 15, 18, 20, 22, 25, 30, 35, 50, 75, 100, 250, 500, or 1000 nucleotides. More preferably the entire amino acid-coding sequence shown in SEQ ID NO:2 is contained within the polynucleotide. In another embodiment the complete cDNA sequence shown in SEQ ID NO:2 is contained within the polynucleotide. If the polynucleotide is an RNA molecule one preferred embodiment is a 6.2 kb transcript.

[0038] Polynucleotides according to the invention contain either the wild type sequence as shown in SEQ ID NO:2 or polymorphisms, such as those shown in Table 2 or Table 3. Other polymorphisms can be readily identified using the method shown below in the examples. Isolated and purified polynucleotides according to the present invention are separated from the sequences to which they are adjacent in the human genome. Thus such isolated polynucleotides comprise less than a full chromosome or other genomic element. Moreover, purified polynucleotides are not present in a mixture of total genomic DNA or a library comprising total genomic DNA or cDNA. Purified polynucleotides have been separated from other sequences, usually by a process such as cloning, hybridization, or amplification such that a population of polynucleotide molecules is predominantly (>50%) the type of molecule which contains the polynucleotide containing BION1 sequences.

[0039] Polynucleotides according to the present invention are conveniently maintained, propagated, and expressed in a vector. The vector can be any useful vector known in the art, whether bacterial or eukaryotic, viral or plasmid. Vectors can be maintained and grown in host cells which are suitable for replication of the particular vector chosen. Appropriate pairs of vectors and host cells are well known in the art. Such host cells carrying BION1 coding sequences can be used to prepare quantities of BION1 protein. Host cells can be grown under suitable growth and expression conditions. BION1 will typically be extracted from the cells, but under certain conditions and with certain genetic constructs, may be isolatable from the culture medium.

[0040] Susceptibility to Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome can be determined according to one aspect of the invention by tracking a polymorphism identified in a proband through other family members. The proband is an affected individual, having either Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome. A nucleotide at a defined location in a BION1 gene of a human is determined to identify whether the test individual contains the polymorphism carried by the proband. The polymorphic nucleotide can be one of the polymorphisms indicated in Tables 2 and 3 or can be a different nucleotide in the same codon, or a different nucleotide in a different codon. The nucleotide at the defined location can be determined by any means known in the art, including but not limited to sequencing, restriction enzyme digestion, allele-specific ligation, allele-specific amplification. The nucleotide at the defined location of the human is compared to that of the affected family member (proband) having a polymorphism at the nucleotide. Such comparison can be by any means known in the art, such as by side-by-side testing, or by serial testing at two different times, with the same or a different technique. The testing and/or comparison can be mannually accomplished or can be done by a machine or computer implemented technique. The human being tested is identified as susceptible to schizophrenia or panic disorder if the determined nucleotide contains the polymorphism found in the affected family member. As is well known in the art, susceptibility does not indicate that there is a 100% probability that the individual will develop the disease symptoms. Other genetic factors and environmental factors are believed to be involved in determining disease elaboration.

[0041] Another method of determining susceptibility to Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome requires knowledge of no other affected family member. Thus the tested patient may be the first in a family to be affected or may have no knowledge of prior affected members, or prior affected family members may not be available for sample donation. A nucleotide at a defined location in a BION1 gene of a human is determined. If the determined nucleotide is a polymorphism identified in Table 2 or Table 3, the human is identified as susceptible to Panic Disorder Syndrome, Bipolar Disorder, or Schizophrenia. As the collection of identified polymorphisms grows, this knowledge can be used to identify susceptibility in others. Thus the list of Table 2 or Table 3 will become more extensive as additional knowledge accumulates regarding other families and other polymorphisms. The expanded list of polymorphisms can be used, like Table 2 or Table 3 as provided herein. Thus Table 2 and Table 3 represent any set of polymorphisms in BION1 previously identified at the time of performing the method.

[0042] Probes and primers for BION1 are also provided which are useful for performing diagnoses according to the invention. Polynucleotides for use as primers or probes typically comprise at least 18 contiguous nucleotides of a human BION1 coding sequence, however, primers and probes of at least 10, 12, 14, 16, 20, 22, or 25 nucleotides may be useful, as well. Polynucleotides encoding only a portion of BION1 can also be fused to other genes to form fusion genes encoding fusion proteins. Preferably the polynucleotide comprises at least one codon identified in Table 1, which codon is for an amino acid found in human BION1 proteins at the corresponding position. More preferably the polynucleotide contains no codons identified in Table 1 as being a rat BION1 codon.

[0043] Human BION1 protein according to the invention is useful inter alia for screening for potential therapeutic agents. Typically such protein is isolated and purified away from whole human cells or recombinant cells, as well as away from other human proteins and possibly also other proteins from a recombinant BION1-producing cell. The BION1 proteins according to the invention typically comprise an amino acid sequence as shown in SEQ ID NO:1. Alternatively the sequence can be that of a polymorphic variant found in Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome patients. More preferably it can be the sequence of a polymorphic variant disclosed in Tables 2 or 3.

[0044] BION1 polypeptides comprise at least 6 amino acids of a human BION1 protein, i.e., enought to form an epitope which can be used to generate antibodies or immune cells which are specific for BION1. The polypeptides can comprise at least 10, 15, 20, 25, 30, 50, or even 75 contiguous amino acids of the BION1 protein. Preferably the polypeptide comprises an amino acid sequence found in humans but not in rats as identified in Table 1. Among other uses, the antibodies to BION1 can be used in binding assays to test substances which may be useful therapeutically. The antibodeies, whether polyclonal or monoclonal, can be used to identify bound or unbound BION1 in an assay or as a competitor molecule for BION1 binding.

[0045] Test substances can be screened for candidates useful in treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome by using the human BION1 protein. It is contacted with a test substance, preferably with a library of test substances serially or simultaneously in separate reaction mixtures. Binding of a test substance to the human BION1 protein is determined. The BION1 protein may be wild-type or polymorphic, including but not limited to one of the polymorphisms disclosed in Tables 2 and 3. Any binding assay known in the art can be used. In some binding assays one of the binding partners is immobilized on a solid support. In other binding assays one binding partner is labeled. In some binding assays competition with a known binder is used as an indicator of binding activity. Those of skill in the art will readily understand how to set up convenient binding assays. A test substance is identified as a candidate drug useful for treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome if it binds to human BION1.

[0046] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1

[0047] Identification of C. elegans Homologs of Mammalian Voltage-Gated Ion Channels.

[0048] Sequences were identified within the C. elegans cosmids c27f2.3 (U40419) and c11d2.6 (AF045640) in a search of the C. elegans genomic database for sequence fragments that resembled voltage gated sodium (NaV) and calcium (CaV) ion channels. These putative, novel genes were approximately 26% identical, 45% conserved (e-value 2e-48) to human voltage-dependent L-type Ca channel (al subunit).

EXAMPLE 2

[0049] Cloning of Human BION1 cDNA.

[0050] These C. elegans sequences displaying similarity to the ion channels were used to identify a homologous human GenBank EST (#590575, derived from a human pancreatic cell line cDNA library.) This was sequenced and found to contain an open reading frame (ORF) encoding part of the human homolog of the C. elegans genes. Further screening of a pancreatic cDNA library led to the isolation of a full-length human cDNA clone (BION1). The complete human cDNA sequence of the human BION1 gene (SEQ ID NO:2) and its conceptual translation product (SEQ ID NO:1) are shown in FIG. 1. The predicted human and rat protein sequences contain 1738 amino acids, and are highly conserved, showing only 24 cross-species amino acid substitutions (1.4%), 12 of which are conservative. The predicted BION1 protein is comprised of four domains (I-IV), each containing six putative membrane-spanning regions (S1-6) and a pore loop (P) between S5 and S6 (FIG. 2a). The predicted topology of the BION1 protein is shown diagrammatically in FIG. 2b. TABLE 1 Amino acid substitutions between human and rat BION1 proteins. Position of first Amino acid Amino acid nucleotide in codon in human in rat Conservative? 1058 Ala Ser Conservative 1241 Ser Thr Conservative 1949 Val Leu Conservative 2069 Val Leu Conservative 2261 Leu Phe 2267 Ser Ile 2276 Thr Ser Conservative 2300 His Asn Conservative 2303 Ser Pro 2312 Ser Pro 2315 Ala Thr 2318 Ile Val Conservative 2909 Ile Leu Conservative 4687 Lys Ser 4670 Arg ? 5117 Asn His Conservative 5120 Ser Asn Conservative 5123 Met Thr Conservative 5135 Thr Ser Conservative 5207 Ala Thr 5228 Phe Ile 5297 Leu Phe 5348 Thr Pro 5354 Ala Thr

EXAMPLE 3

[0051] Cloning of Rat BION1 cDNA.

[0052] The human BION1 cDNA was used to screen a rat brain cDNA library in order to isolate a full-length rat BION1 channel cDNA. At the amino-acid level, this protein exhibited 98.6% identity to the existing human sequence. Recently, another group has reported cloning and sequencing the rat BION1 gene (AF078779) (Lee et al. 1999.)

EXAMPLE 4

[0053] BION1 Protein Sequence is Divergent from Other Voltage-Gated Ion Channels.

[0054] Sequence alignments of BION1 with other voltage gated ion channels indicate significant divergence. Amino-acid residues thought crucial for determining ion selectivity are in a novel arrangement. BION1 has EEKE selectivity amino acids in its pore loop region, in contrast to most mammalian Ca_(V) channels with EEEE at these positions, and Na_(V) with DEKA (FIG. 3a). Cladistic analysis of BION1 and its C. elegans homologs suggests that these comprise a distinct family of channels that diverged from Ca_(V) and Na_(V) before the duplication that gave rise to these two more closely related families (FIG. 3b). BION1 family members have a reduced number of positively charged amino-acid residues in the S4 regions, which are believed to act as part of the voltage-sensing apparatus in other families of voltage-dependent ion channels. This reduction in number of charges is particularly pronounced in domain IV, and is suggestive that the activation of this channel may be relatively slow compared to Ca_(V) and Na_(V). BION1 channels have a divergent domain III-IV linker, missing the amino-acid sequence IFM, which has been shown to be important in mammalian Na_(V) channel inactivation. This suggests that BION1 channel inactivation may be slow compared to Na_(V).

EXAMPLE 5

[0055] Expression of BION1.

[0056] Northern analyses were performed using the rat BION1 gene. BION1 was found to be expressed abundantly in rat brain, spinal cord, and pancreas as a ˜6.2 kb transcript (FIG. 4a). Significant expression is also seen in heart, kidney, small intestine and placenta. The expression in heart is of significance given the mitral valve prolapse phenotype of Panic Disorder Syndrome.

[0057] In the brain, BION1 is expressed most highly in the thalamus, but is also expressed in other regions of the brain (FIG. 4b). This distribution has been confirmed by RT-PCR. In situ hybridization using BION1 cRNA shows primarily neuronal staining, with strong hybridization to the olfactory bulb, piriform cortex, hippocampal neuronal layers, suprachiasmatic nucleus, medial habenular nucleus, locus coeruleus, and subformical organ, and at low levels throughout the CNS (FIG. 5).

EXAMPLE 6

[0058] Chromosomal Mapping of BION1.

[0059] A BAC clone (2294N13) was shown to contain BION1 by PCR analysis and sequencing using BION1 primers. FISH analysis of the BION1 BAC clone in the Ward laboratory (Yale University) localized the human gene to human chromosomal band 13q32 (FIG. 6).

[0060] A well-defined schizophrenia susceptibility locus (SCZD7) is located on 13q32 (Lin et al. 1995, Blouin et al. 1998). The genetic linkage marker with the highest LOD score was CHLC.ATA26D07 (D13S779)(Blouin et al. 1998). A second neuropsychiatric disorder, Panic Disorder, was also linked to the D13S779 marker (Weissman et al. 2000).

[0061] A YAC contig covering the 13q32 region including and surrounding the D13S779 marker was developed in the Ward laboratory and 2 YACs from the contig were demonstrated to contain the BION1 gene by PCR analysis using BION1 cDNA primers. The 2 YACs (968g12 and 761a1) share a single STS marker, D13S919, which is immediately adjacent to D13S779 on the MIT genetic linkage map. D13S779 is found only in YAC 761al. Thus BION1 physically maps to the same YAC as D13S779, the genetic marker with the highest LOD score with the 13q32 schizophrenia susceptibility locus.

EXAMPLE 7

[0062] Genomic Structure of BION1.

[0063] In order to determine the genomic structure of BION1, the BION1 BAC clone was sequenced in its entirety and additional overlapping BACs were identified, both by screening various BAC libraries with the available ESTs, and by database analysis. Genomic sequence of a second BION1-containing BAC, RP11-45P5, was recently completed as part of the Human Genome Project (Birren et al., unpublished). Together, these BAC sequences have allowed us to completely define the genomic structure of BION1. The BION1 gene is composed of 42 exons (FIG. 7). The 3′ portion of this gene (corresponding to bp 1855-6942 of the BION1 cDNA) spans 175 kb of genomic DNA; sequencing of the 5′ introns is as yet incomplete. However, by analysis of sequence of the two BAC clones, and by genomic PCR and sequencing, intron-exon boundaries and intronic sequences flanking individual exons have been obtained for all 42 exons.

EXAMPLE 8

[0064] Evidence Linking BION1 to SCZD7.

[0065] Genetic co-localization of BION1 and SCZD7, together with congruent tissue distribution and predicted function of the BION1 protein, constitutes strong but indirect evidence that the BION1 gene is the 13q32 schizophrenia susceptibility gene SCZD7. In order to validate this hypothesis, mutational analysis of BION1 was undertaken in cohorts of schizophrenia and Panic Disorder Syndrome patients.

[0066] Approach to Identify Mutations in BION1.

[0067] Given the polygenic nature of schizophrenia susceptibility, sequencing of the BION1 exons from the genomic DNA of large numbers of unrelated patient probands (48 Panic Disorder and 48 Schizophrenic patient probands) was undertaken in an attempt to identify mutations.

[0068] Panic Disorder Patient Selection.

[0069] We were fortunate to have access to the same cohort that Weissman et al. (2000) used to map Panic Disorder Syndrome to 13q32. Families were initially accepted into this study if at least 3 relatives appeared affected by panic disorder. A detailed diagnosis and pedigree analysis were then performed, and the data blinded to name and family. Three senior clinical investigators independently diagnosed each individual, and any case that had a discrepant diagnosis among the physicians was flagged for additional data collection and review. Since these samples were closely linked to 13q32, it was expected that all of these patients would be defective at 13q32.

[0070] Schizophrenia Patient Selection.

[0071] Genomic DNA from DSM-III-R criteria (Diagnostic and Statistical Manual of Mental Disorders) schizophrenic patient probands was obtained from the NIMH Schizophrenia Genetics Initiative collection. Samples are included in this collection if several conditions are met. Firstly, at least two affected individuals must be biologically related as first-degree relatives diagnosed with DSM-III-R schizophrenia (SZ) or schizoaffective disorder depressive type (SADD). If this condition is met, a pedigree is established and extended. Two senior psychiatrists or clinical psychologists separately and independently assess data from clinical interviews and family history to arrive at a diagnosis. In the case of a disagreement, a third senior psychiatrist or clinical psychologist reviewed all available data and a written summary of the discrepant points of view. The third clinician acts as a tie-breaker. Since there are at least 9 schizophrenia loci, it was expected that about 10% of these patients would be defective at SCZD7.

[0072] Method of Polymorphism ID.

[0073] Polymorphisms were initially detected by sequencing the relevant exon. Sequencing in the second direction was performed routinely for all exonic regions. In addition, the redundancy of the study, sequencing 48 schizophrenia and 48 Panic Disorder Syndrome samples, assures that the same polymorphism is detected in multiple samples. We did not systematically include control DNA, reasoning that most exons would be normal at most positions, and that a mutation at on position would be evident on comparison to the BION1 cDNA sequence.

[0074] Exons Scanned for Polymorphisms in Panic Disorder Syndrome Samples.

[0075] A total of 13 exons have been scanned, as well as the 3′ UTR, for 48 Panic Disorder Syndrome samples. This represents 1405 bp out of the 5231 bp coding sequence (27%) and 3050 bp out of the 6942 bp cDNA (44%). We found 2 exonic, 3 intronic, and 4 3′UTR polymorphisms (Table 2) (see FIG. 8). TABLE 2 Polymorphisms found in Panic Disorder Syndrome gDNA samples Allele Frequency Exon Polymorphism ID A/A* A/B* B/B* C3 T(−251)A† 2 14 12 T(−221)C† 12 14 2 C13 G(−33)A† 10 21 9 C10 C1822T 24 13 11 C17 A2065G 39 4 0 3′UTR G6355A 3 4 4 T6370C 11 11 21 C6442T 24 10 10 G6453A 43 0 1

[0076] Exons Scanned for Polymorphisms in Schizophrenia Samples.

[0077] The same 13 exons were scanned with the 48 Schizophrenia patient DNAs. This represents 1405 bp out of the 5231 bp coding sequence (27%). We found the same 2 exonic polymorphisms seen in the Panic Disorder samples, plus one novel polymorphism not seen in the previous scan (Table 3) (see FIG. 8). TABLE 3 Polymorphisms found in NIMH Schizophrenic gDNA samples Allele Frequency Exon Polymorphism ID A/A* A/B* B/B* C13 C1102T 32 1 0 C10 C1822T 28 18 1 C17 A2065G 34 3 0

[0078] Polymorphisms Identified.

[0079] A2065G was found to be heterozygous in 3 schizophrenia samples (Repository IDs 90C00517, 90C03310, 90C03592) out of 37 (8%). This polymorphism was present in 4/43 (9.5%) of Panic Disorder Syndrome samples, so this polymorphism is consistently rare in both disorders. Interestingly, this consistency was not true when considering C1822T. In schizophrenia samples, this polymorphic nucleotide is T in only 1 sample (Repository ID 90C00518) out of 47 (2%). When the same polymorphism is examined in Panic Disorder Syndrome patients, {fraction (11/48)} (23%) show a T at this position. The C1822T and A2065G polymorphisms were confirmed by sequencing the relevant exon in both directions. The intronic and 5′UTR polymorphisms were sequenced in Panic Disorder Syndrome samples, and not schizophrenia samples. C1102T was only seen in 1 patient with schizophrenia (Repository ID 90C02418), and no Panic Disorder Syndrome samples. Although this difference was seen in a rerun of the same sample, sequencing in the second direction was not done, so the identification of this polymorphism is tentative.

[0080] Confirmation of Polymorphisms.

[0081] In cases where the polymorphism was found to have generated or destroyed a restriction endonuclease cleavage site, then the existence of the polymorphism could be confirmed by performing a restriction digest on the relevant PCR product. This was the case for C1822T, where the polymorphic nucleotide led to the destruction of a TaqI site. TaqI digestion and agarose gel analysis confirmed the prediction that sequence analysis allowed us to make.

REFERENCES

[0082] Andreasen, N. C. (1995) Symptoms, signs, and diagnosis of schizophrenia. Lancet 346:477-481.

[0083] Blouin, J-L et al. (1998) Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21. Nature Genetics 20:70-73.

[0084] Brzustowicz, L. M. et al. (2000) Location of a Major Susceptibility Locus for Familial Schizophrenia on Chromosome 1q21-q22. Science 288:678-682

[0085] Knowles et al. (1998) Results of a genome-wide screen for panic disorder. Am J Med Genet (Neuropsychiatr Genet) 81:139-147.

[0086] Lee, J-H et al. (1999) Cloning of a novel four repeat protein related to voltage-gated sodium and calcium channels. FEBS Letters 445:231-236.

[0087] Millar, J. K. et al. (2000) Disruption of two novel genes by a translocation co-segregating with schizophrenia. Hum Mol Genet. 9:1415-1423.

[0088] Weissman, M. S et al. (2000) Potential Panic Disorder Syndrome: Clinical and Genetic Linkage Evidence. American Journal of Medical Genetics (Neuropsychiatric Genetics) 96:24-35

1 3 1 1738 PRT Homo sapiens 1 Met Leu Lys Arg Lys Gln Ser Ser Arg Val Glu Ala Gln Pro Val Thr 1 5 10 15 Asp Phe Gly Pro Asp Glu Ser Leu Ser Asp Asn Ala Asp Ile Leu Trp 20 25 30 Ile Asn Lys Pro Trp Val His Ser Leu Leu Arg Ile Cys Ala Ile Ile 35 40 45 Ser Val Ile Ser Val Cys Met Asn Thr Pro Met Thr Phe Glu His Tyr 50 55 60 Pro Pro Leu Gln Tyr Val Thr Phe Thr Leu Asp Thr Leu Leu Met Phe 65 70 75 80 Leu Tyr Thr Ala Glu Met Ile Ala Lys Met His Ile Arg Gly Ile Val 85 90 95 Lys Gly Asp Ser Ser Tyr Val Lys Asp Arg Trp Cys Val Phe Asp Gly 100 105 110 Phe Met Val Phe Cys Leu Trp Val Ser Leu Val Leu Gln Val Phe Glu 115 120 125 Ile Ala Asp Ile Val Asp Gln Met Ser Pro Trp Gly Met Leu Arg Ile 130 135 140 Pro Arg Pro Leu Ile Met Ile Arg Ala Phe Arg Ile Tyr Phe Arg Phe 145 150 155 160 Glu Leu Pro Arg Thr Arg Ile Thr Asn Ile Leu Lys Arg Ser Gly Glu 165 170 175 Gln Ile Trp Ser Val Ser Ile Phe Leu Leu Phe Phe Leu Leu Leu Tyr 180 185 190 Gly Ile Leu Gly Val Gln Met Phe Gly Thr Phe Thr Tyr His Cys Val 195 200 205 Val Asn Asp Thr Lys Pro Gly Asn Val Thr Trp Asn Ser Leu Ala Ile 210 215 220 Pro Asp Thr His Cys Ser Pro Glu Leu Glu Glu Gly Tyr Gln Cys Pro 225 230 235 240 Pro Gly Phe Lys Cys Met Asp Leu Glu Asp Leu Gly Leu Ser Arg Gln 245 250 255 Glu Leu Gly Tyr Ser Gly Phe Asn Glu Ile Gly Thr Ser Ile Phe Thr 260 265 270 Val Tyr Glu Ala Ala Ser Gln Glu Gly Trp Val Phe Leu Met Tyr Arg 275 280 285 Ala Ile Asp Ser Phe Pro Arg Trp Arg Ser Tyr Phe Tyr Phe Ile Thr 290 295 300 Leu Ile Phe Phe Leu Ala Trp Leu Val Lys Asn Val Phe Ile Ala Val 305 310 315 320 Ile Ile Glu Thr Phe Ala Glu Ile Arg Val Gln Phe Gln Gln Met Trp 325 330 335 Gly Ser Arg Ser Ser Thr Thr Ser Thr Ala Thr Thr Gln Met Phe His 340 345 350 Glu Asp Ala Ala Gly Gly Trp Gln Leu Val Ala Val Asp Val Asn Lys 355 360 365 Pro Gln Gly Arg Ala Pro Ala Cys Leu Gln Lys Met Met Arg Ser Ser 370 375 380 Val Phe His Met Phe Ile Leu Ser Met Val Thr Val Asp Val Ile Val 385 390 395 400 Ala Ala Ser Asn Tyr Tyr Lys Gly Glu Asn Phe Arg Arg Gln Tyr Asp 405 410 415 Glu Phe Tyr Leu Ala Glu Val Ala Phe Thr Val Leu Phe Asp Leu Glu 420 425 430 Ala Leu Leu Lys Ile Trp Cys Leu Gly Phe Thr Gly Tyr Ile Ser Ser 435 440 445 Ser Leu His Lys Phe Glu Leu Leu Leu Val Ile Gly Thr Thr Leu His 450 455 460 Val Tyr Pro Asp Leu Tyr His Ser Gln Phe Thr Tyr Phe Gln Val Leu 465 470 475 480 Arg Val Val Arg Leu Ile Lys Ile Ser Pro Ala Leu Glu Asp Phe Val 485 490 495 Tyr Lys Ile Phe Gly Pro Gly Lys Lys Leu Gly Ser Leu Val Val Phe 500 505 510 Thr Ala Ser Leu Leu Ile Val Met Ser Ala Ile Ser Leu Gln Met Phe 515 520 525 Cys Phe Val Glu Glu Leu Asp Arg Phe Thr Thr Phe Pro Arg Ala Phe 530 535 540 Met Ser Met Phe Gln Ile Leu Thr Gln Glu Gly Trp Val Asp Val Met 545 550 555 560 Asp Gln Thr Leu Asn Ala Val Gly His Met Trp Ala Pro Val Val Ala 565 570 575 Ile Tyr Phe Ile Leu Tyr His Leu Phe Ala Thr Leu Ile Leu Leu Ser 580 585 590 Leu Phe Val Ala Val Ile Leu Asp Asn Leu Glu Leu Asp Glu Asp Leu 595 600 605 Lys Lys Leu Lys Gln Leu Lys Gln Ser Glu Ala Asn Ala Asp Thr Lys 610 615 620 Glu Lys Leu Pro Leu Arg Leu Arg Ile Phe Glu Lys Phe Pro Asn Arg 625 630 635 640 Pro Gln Met Val Lys Ile Ser Lys Leu Pro Ser Asp Phe Thr Val Pro 645 650 655 Lys Ile Arg Glu Ser Phe Met Lys Gln Phe Ile Asp Arg Gln Gln Gln 660 665 670 Asp Thr Cys Cys Leu Leu Arg Ser Leu Pro Thr Thr Ser Ser Ser Ser 675 680 685 Cys Asp His Ser Lys Arg Ser Ala Ile Glu Asp Asn Lys Tyr Ile Asp 690 695 700 Gln Lys Leu Arg Lys Ser Val Phe Ser Ile Arg Ala Arg Asn Leu Leu 705 710 715 720 Glu Lys Glu Thr Ala Val Thr Lys Ile Leu Arg Ala Cys Thr Arg Gln 725 730 735 Arg Met Leu Ser Gly Ser Phe Glu Gly Gln Pro Ala Lys Glu Arg Ser 740 745 750 Ile Leu Ser Val Gln His His Ile Arg Gln Glu Arg Arg Ser Leu Arg 755 760 765 His Gly Ser Asn Ser Gln Arg Ile Ser Arg Gly Lys Ser Leu Glu Thr 770 775 780 Leu Thr Gln Asp His Ser Asn Thr Val Arg Tyr Arg Asn Ala Gln Arg 785 790 795 800 Glu Asp Ser Glu Ile Lys Met Ile Gln Glu Lys Lys Glu Gln Ala Glu 805 810 815 Met Lys Arg Lys Val Gln Glu Glu Glu Leu Arg Glu Asn His Pro Tyr 820 825 830 Phe Asp Lys Pro Leu Phe Ile Val Gly Arg Glu His Arg Phe Arg Asn 835 840 845 Phe Cys Arg Val Val Val Arg Ala Arg Phe Asn Ala Ser Lys Thr Asp 850 855 860 Pro Val Thr Gly Ala Val Lys Asn Thr Lys Tyr His Gln Leu Tyr Asp 865 870 875 880 Leu Leu Gly Leu Val Thr Tyr Leu Asp Trp Val Met Ile Ile Val Thr 885 890 895 Ile Cys Ser Cys Ile Ser Met Met Phe Glu Ser Pro Phe Arg Arg Val 900 905 910 Met His Ala Pro Thr Leu Gln Ile Ala Glu Tyr Val Phe Val Ile Phe 915 920 925 Met Ser Ile Glu Leu Asn Leu Lys Ile Met Ala Asp Gly Leu Phe Phe 930 935 940 Thr Pro Thr Ala Val Ile Arg Asp Phe Gly Gly Val Met Asp Ile Phe 945 950 955 960 Ile Tyr Leu Val Ser Leu Ile Phe Leu Cys Trp Met Pro Gln Asn Val 965 970 975 Pro Ala Glu Ser Gly Ala Gln Leu Leu Met Val Leu Arg Cys Leu Arg 980 985 990 Pro Leu Arg Ile Phe Lys Leu Val Pro Gln Met Arg Lys Val Val Arg 995 1000 1005 Glu Leu Phe Ser Gly Phe Lys Glu Ile Phe Leu Val Ser Ile Leu Leu 1010 1015 1020 Leu Thr Leu Met Leu Val Phe Ala Ser Phe Gly Val Gln Leu Phe Ala 1025 1030 1035 1040 Gly Lys Leu Ala Lys Cys Asn Asp Pro Asn Ile Ile Arg Arg Glu Asp 1045 1050 1055 Cys Asn Gly Ile Phe Arg Ile Asn Val Ser Val Ser Lys Asn Leu Asn 1060 1065 1070 Leu Lys Leu Arg Pro Gly Glu Lys Lys Pro Gly Phe Trp Val Pro Arg 1075 1080 1085 Val Trp Ala Asn Pro Arg Asn Phe Asn Phe Asp Asn Val Gly Asn Ala 1090 1095 1100 Met Leu Ala Leu Phe Glu Val Leu Ser Leu Lys Gly Trp Val Glu Val 1105 1110 1115 1120 Arg Asp Val Ile Ile His Arg Val Gly Pro Ile His Gly Ile Tyr Ile 1125 1130 1135 His Val Phe Val Phe Leu Gly Cys Met Ile Gly Leu Thr Leu Phe Val 1140 1145 1150 Gly Val Val Ile Ala Asn Phe Asn Glu Asn Lys Gly Thr Ala Leu Leu 1155 1160 1165 Thr Val Asp Gln Arg Arg Trp Glu Asp Leu Lys Ser Arg Leu Lys Ile 1170 1175 1180 Ala Gln Pro Leu His Leu Pro Pro Arg Pro Asp Asn Asp Gly Phe Arg 1185 1190 1195 1200 Ala Lys Met Tyr Asp Ile Thr Gln His Pro Phe Phe Lys Arg Thr Ile 1205 1210 1215 Ala Leu Leu Val Leu Ala Gln Ser Val Leu Leu Ser Val Lys Trp Asp 1220 1225 1230 Val Glu Asp Pro Val Thr Val Pro Leu Ala Thr Met Ser Val Val Phe 1235 1240 1245 Thr Phe Ile Phe Val Leu Glu Val Thr Met Lys Ile Ile Ala Met Ser 1250 1255 1260 Pro Ala Gly Phe Trp Gln Ser Arg Arg Asn Arg Tyr Asp Leu Leu Val 1265 1270 1275 1280 Thr Ser Leu Gly Val Val Trp Val Val Leu His Phe Ala Leu Leu Asn 1285 1290 1295 Ala Tyr Thr Tyr Met Met Gly Ala Cys Val Ile Val Phe Arg Phe Phe 1300 1305 1310 Ser Ile Cys Gly Lys His Val Thr Leu Lys Met Leu Leu Leu Thr Val 1315 1320 1325 Val Val Ser Met Tyr Lys Ser Phe Phe Ile Ile Val Gly Met Phe Leu 1330 1335 1340 Leu Leu Leu Cys Tyr Ala Phe Ala Gly Val Val Leu Phe Gly Thr Val 1345 1350 1355 1360 Lys Tyr Gly Glu Asn Ile Asn Arg His Ala Asn Phe Ser Ser Ala Gly 1365 1370 1375 Lys Ala Ile Thr Val Leu Phe Arg Ile Val Thr Gly Glu Asp Trp Asn 1380 1385 1390 Lys Ile Met His Asp Cys Met Val Gln Pro Pro Phe Cys Thr Pro Asp 1395 1400 1405 Glu Phe Thr Tyr Trp Ala Thr Asp Cys Gly Asn Tyr Ala Gly Ala Leu 1410 1415 1420 Met Tyr Phe Cys Ser Phe Tyr Val Ile Ile Ala Tyr Ile Met Leu Asn 1425 1430 1435 1440 Leu Leu Val Ala Ile Ile Val Glu Asn Phe Ser Leu Phe Tyr Ser Thr 1445 1450 1455 Glu Glu Asp Gln Leu Leu Ser Tyr Asn Asp Leu Arg His Phe Gln Ile 1460 1465 1470 Ile Trp Asn Met Val Asp Asp Lys Arg Glu Gly Val Ile Pro Thr Phe 1475 1480 1485 Arg Val Lys Phe Leu Leu Arg Leu Leu Arg Gly Arg Leu Glu Val Asp 1490 1495 1500 Leu Asp Lys Asp Lys Leu Leu Phe Lys His Met Cys Tyr Glu Met Glu 1505 1510 1515 1520 Arg Leu His Asn Gly Gly Asp Val Thr Phe His Asp Val Leu Ser Met 1525 1530 1535 Leu Ser Tyr Arg Ser Val Asp Ile Arg Lys Ser Leu Gln Leu Glu Glu 1540 1545 1550 Leu Leu Ala Arg Glu Gln Leu Glu Tyr Thr Ile Glu Glu Glu Val Ala 1555 1560 1565 Lys Gln Thr Ile Arg Met Trp Leu Lys Lys Cys Leu Lys Arg Ile Arg 1570 1575 1580 Ala Lys Gln Gln Gln Ser Cys Ser Ile Ile His Ser Leu Arg Glu Ser 1585 1590 1595 1600 Gln Gln Gln Glu Leu Ser Arg Phe Leu Asn Pro Pro Ser Ile Glu Thr 1605 1610 1615 Thr Gln Pro Ser Glu Asp Thr Asn Ala Asn Ser Gln Asp Asn Ser Met 1620 1625 1630 Gln Pro Glu Thr Ser Ser Gln Gln Gln Leu Leu Ser Pro Thr Leu Ser 1635 1640 1645 Asp Arg Gly Gly Ser Arg Gln Asp Ala Ala Asp Ala Gly Lys Pro Gln 1650 1655 1660 Arg Lys Phe Gly Gln Trp Arg Leu Pro Ser Ala Pro Lys Pro Ile Ser 1665 1670 1675 1680 His Ser Val Ser Ser Val Asn Leu Arg Leu Gly Gly Arg Thr Thr Met 1685 1690 1695 Lys Ser Val Val Cys Lys Met Asn Pro Met Thr Asp Ala Ala Ser Cys 1700 1705 1710 Gly Ser Glu Val Lys Lys Trp Trp Thr Arg Gln Leu Thr Val Glu Ser 1715 1720 1725 Asp Glu Ser Gly Asp Asp Leu Leu Asp Ile 1730 1735 2 6952 DNA Homo sapiens CDS (230)...(5443) 2 ggcacgagcg agcgtgagcc gcggccccag ccgggccgag cgcgctgcct gagctgagcc 60 gccgtaggtg aggggcccgc gtccccgccc gccctgggcg ccgcgcctgg cactgatcct 120 gccggtcgcc cactgtcgcc gccgccgccg cccgcgggca ccatgacagc tctgagcgct 180 ggggttacag actgtggttt tgtgcttgct caccaaagct aacctcagc atg ctc aaa 238 Met Leu Lys 1 agg aag cag agt tcc agg gtg gaa gcc cag cca gtc act gac ttt ggt 286 Arg Lys Gln Ser Ser Arg Val Glu Ala Gln Pro Val Thr Asp Phe Gly 5 10 15 cct gat gag tct ctg tcg gat aat gct gac atc ctc tgg att aac aaa 334 Pro Asp Glu Ser Leu Ser Asp Asn Ala Asp Ile Leu Trp Ile Asn Lys 20 25 30 35 cca tgg gtt cac tct ttg ctg cgc atc tgt gcc atc atc agc gtc att 382 Pro Trp Val His Ser Leu Leu Arg Ile Cys Ala Ile Ile Ser Val Ile 40 45 50 tct gtt tgt atg aat acg cca atg acc ttc gag cac tat cct cca ctt 430 Ser Val Cys Met Asn Thr Pro Met Thr Phe Glu His Tyr Pro Pro Leu 55 60 65 cag tat gtg acc ttc act ttg gat aca tta ttg atg ttt ctc tac acg 478 Gln Tyr Val Thr Phe Thr Leu Asp Thr Leu Leu Met Phe Leu Tyr Thr 70 75 80 gca gag atg ata gca aaa atg cac atc cgg ggc att gtc aag ggg gat 526 Ala Glu Met Ile Ala Lys Met His Ile Arg Gly Ile Val Lys Gly Asp 85 90 95 agt tcc tat gtg aaa gat cgc tgg tgt gtt ttt gat gga ttt atg gtc 574 Ser Ser Tyr Val Lys Asp Arg Trp Cys Val Phe Asp Gly Phe Met Val 100 105 110 115 ttt tgc ctt tgg gtt tct ttg gtg cta cag gtg ttt gaa att gct gat 622 Phe Cys Leu Trp Val Ser Leu Val Leu Gln Val Phe Glu Ile Ala Asp 120 125 130 ata gtt gat cag atg tca cct tgg ggc atg ttg cgg att cca cgg cca 670 Ile Val Asp Gln Met Ser Pro Trp Gly Met Leu Arg Ile Pro Arg Pro 135 140 145 ctg att atg atc cga gca ttc cgg att tat ttc cga ttt gaa ctg cca 718 Leu Ile Met Ile Arg Ala Phe Arg Ile Tyr Phe Arg Phe Glu Leu Pro 150 155 160 agg acc aga att aca aat att tta aag cga tcg gga gaa caa ata tgg 766 Arg Thr Arg Ile Thr Asn Ile Leu Lys Arg Ser Gly Glu Gln Ile Trp 165 170 175 agt gtt tcc att ttt cta ctt ttc ttt cta ctt ctt tat gga att tta 814 Ser Val Ser Ile Phe Leu Leu Phe Phe Leu Leu Leu Tyr Gly Ile Leu 180 185 190 195 gga gtt cag atg ttt gga aca ttt act tat cac tgt gtt gta aat gac 862 Gly Val Gln Met Phe Gly Thr Phe Thr Tyr His Cys Val Val Asn Asp 200 205 210 aca aag cca ggg aat gta acc tgg aat agt tta gct att cca gac aca 910 Thr Lys Pro Gly Asn Val Thr Trp Asn Ser Leu Ala Ile Pro Asp Thr 215 220 225 cac tgc tca cca gag cta gaa gaa ggc tac cag tgc cca cct gga ttt 958 His Cys Ser Pro Glu Leu Glu Glu Gly Tyr Gln Cys Pro Pro Gly Phe 230 235 240 aaa tgc atg gac ctt gaa gat ctg gga ctt agc agg caa gag ctg ggc 1006 Lys Cys Met Asp Leu Glu Asp Leu Gly Leu Ser Arg Gln Glu Leu Gly 245 250 255 tac agt ggc ttt aat gag ata gga act agt ata ttc acc gtc tat gag 1054 Tyr Ser Gly Phe Asn Glu Ile Gly Thr Ser Ile Phe Thr Val Tyr Glu 260 265 270 275 gcc gcc tca cag gaa ggc tgg gtg ttc ctc atg tac aga gca att gac 1102 Ala Ala Ser Gln Glu Gly Trp Val Phe Leu Met Tyr Arg Ala Ile Asp 280 285 290 agc ttt ccc cgt tgg cgt tcc tac ttc tat ttc atc act ctc att ttc 1150 Ser Phe Pro Arg Trp Arg Ser Tyr Phe Tyr Phe Ile Thr Leu Ile Phe 295 300 305 ttc ctc gcc tgg ctt gtg aag aac gtg ttt att gct gtt atc att gaa 1198 Phe Leu Ala Trp Leu Val Lys Asn Val Phe Ile Ala Val Ile Ile Glu 310 315 320 aca ttt gca gaa atc aga gta cag ttt caa caa atg tgg gga tcg aga 1246 Thr Phe Ala Glu Ile Arg Val Gln Phe Gln Gln Met Trp Gly Ser Arg 325 330 335 agc agc act act tca aca gcc acc acc cag atg ttt cat gaa gat gct 1294 Ser Ser Thr Thr Ser Thr Ala Thr Thr Gln Met Phe His Glu Asp Ala 340 345 350 355 gct gga ggt tgg cag ctg gta gct gtg gat gtc aac aag ccc cag gga 1342 Ala Gly Gly Trp Gln Leu Val Ala Val Asp Val Asn Lys Pro Gln Gly 360 365 370 cgc gcc cca gcc tgc ctc cag aaa atg atg cgg tca tcc gtt ttc cac 1390 Arg Ala Pro Ala Cys Leu Gln Lys Met Met Arg Ser Ser Val Phe His 375 380 385 atg ttc atc ctg agc atg gtg acc gtg gac gtg atc gtg gcg gct agc 1438 Met Phe Ile Leu Ser Met Val Thr Val Asp Val Ile Val Ala Ala Ser 390 395 400 aac tac tac aaa gga gaa aac ttc agg agg cag tac gac gag ttc tac 1486 Asn Tyr Tyr Lys Gly Glu Asn Phe Arg Arg Gln Tyr Asp Glu Phe Tyr 405 410 415 ctg gcg gag gtg gct ttt aca gta ctt ttt gat ttg gaa gca ctt ctg 1534 Leu Ala Glu Val Ala Phe Thr Val Leu Phe Asp Leu Glu Ala Leu Leu 420 425 430 435 aag ata tgg tgt ttg gga ttt act gga tat att agc tca tct ctc cac 1582 Lys Ile Trp Cys Leu Gly Phe Thr Gly Tyr Ile Ser Ser Ser Leu His 440 445 450 aaa ttc gaa cta cta ctc gta att gga act act ctt cat gta tac cca 1630 Lys Phe Glu Leu Leu Leu Val Ile Gly Thr Thr Leu His Val Tyr Pro 455 460 465 gat ctt tat cat tca caa ttc acg tac ttt cag gtt ctc cga gta gtt 1678 Asp Leu Tyr His Ser Gln Phe Thr Tyr Phe Gln Val Leu Arg Val Val 470 475 480 cgg ctg att aag att tca cct gca tta gaa gac ttt gtg tac aag ata 1726 Arg Leu Ile Lys Ile Ser Pro Ala Leu Glu Asp Phe Val Tyr Lys Ile 485 490 495 ttt ggt cct gga aaa aag ctt ggg agt ttg gtt gta ttt act gcc agc 1774 Phe Gly Pro Gly Lys Lys Leu Gly Ser Leu Val Val Phe Thr Ala Ser 500 505 510 515 ctc ttg att gtt atg tca gca att agt ttg cag atg ttc tgc ttt gtc 1822 Leu Leu Ile Val Met Ser Ala Ile Ser Leu Gln Met Phe Cys Phe Val 520 525 530 gaa gaa ctg gac aga ttt act acg ttt ccg agg gca ttt atg tcc atg 1870 Glu Glu Leu Asp Arg Phe Thr Thr Phe Pro Arg Ala Phe Met Ser Met 535 540 545 ttc cag atc ctc acc cag gaa gga tgg gtg gac gta atg gac caa act 1918 Phe Gln Ile Leu Thr Gln Glu Gly Trp Val Asp Val Met Asp Gln Thr 550 555 560 cta aat gct gtg gga cat atg tgg gca ccc gtg gtt gcc atc tat ttc 1966 Leu Asn Ala Val Gly His Met Trp Ala Pro Val Val Ala Ile Tyr Phe 565 570 575 att ctc tat cat ctt ttt gcc act ctg atc ctc ctg agt ttg ttt gtt 2014 Ile Leu Tyr His Leu Phe Ala Thr Leu Ile Leu Leu Ser Leu Phe Val 580 585 590 595 gct gtt att ttg gac aac tta gaa ctt gat gaa gac cta aag aag ctt 2062 Ala Val Ile Leu Asp Asn Leu Glu Leu Asp Glu Asp Leu Lys Lys Leu 600 605 610 aaa caa tta aag caa agt gaa gca aat gcg gac acc aaa gaa aag ctc 2110 Lys Gln Leu Lys Gln Ser Glu Ala Asn Ala Asp Thr Lys Glu Lys Leu 615 620 625 cct tta cgc ctg cga atc ttt gaa aaa ttt cca aac aga cct caa atg 2158 Pro Leu Arg Leu Arg Ile Phe Glu Lys Phe Pro Asn Arg Pro Gln Met 630 635 640 gtg aaa atc tca aag ctt cct tca gat ttt aca gtt cct aaa atc agg 2206 Val Lys Ile Ser Lys Leu Pro Ser Asp Phe Thr Val Pro Lys Ile Arg 645 650 655 gag agt ttt atg aag cag ttt att gac cgc cag caa cag gac aca tgt 2254 Glu Ser Phe Met Lys Gln Phe Ile Asp Arg Gln Gln Gln Asp Thr Cys 660 665 670 675 tgc ctc ctg aga agc ctc ccg acc acc tct tcc tcc tcc tgc gac cac 2302 Cys Leu Leu Arg Ser Leu Pro Thr Thr Ser Ser Ser Ser Cys Asp His 680 685 690 tcc aaa cgc tca gca att gag gac aac aaa tac atc gac caa aaa ctt 2350 Ser Lys Arg Ser Ala Ile Glu Asp Asn Lys Tyr Ile Asp Gln Lys Leu 695 700 705 cgc aag tct gtt ttc agc atc agg gca agg aac ctt ctg gaa aag gag 2398 Arg Lys Ser Val Phe Ser Ile Arg Ala Arg Asn Leu Leu Glu Lys Glu 710 715 720 acc gca gtc act aaa atc tta aga gct tgc acc cga cag cgc atg ctg 2446 Thr Ala Val Thr Lys Ile Leu Arg Ala Cys Thr Arg Gln Arg Met Leu 725 730 735 agc gga tca ttt gag ggg cag ccc gca aag gag agg tca atc ctc agc 2494 Ser Gly Ser Phe Glu Gly Gln Pro Ala Lys Glu Arg Ser Ile Leu Ser 740 745 750 755 gtg cag cat cat atc cgc caa gag cgc agg tca cta aga cat gga tca 2542 Val Gln His His Ile Arg Gln Glu Arg Arg Ser Leu Arg His Gly Ser 760 765 770 aac agc cag agg atc agc agg gga aaa tct ctt gaa act ttg act caa 2590 Asn Ser Gln Arg Ile Ser Arg Gly Lys Ser Leu Glu Thr Leu Thr Gln 775 780 785 gat cat tcc aat aca gtg aga tat aga aat gca caa aga gaa gac agt 2638 Asp His Ser Asn Thr Val Arg Tyr Arg Asn Ala Gln Arg Glu Asp Ser 790 795 800 gaa ata aag atg att cag gaa aaa aag gag caa gca gag atg aaa agg 2686 Glu Ile Lys Met Ile Gln Glu Lys Lys Glu Gln Ala Glu Met Lys Arg 805 810 815 aaa gtg caa gaa gag gaa ctc aga gag aac cac cca tac ttc gat aag 2734 Lys Val Gln Glu Glu Glu Leu Arg Glu Asn His Pro Tyr Phe Asp Lys 820 825 830 835 cca ctg ttc att gtc ggg cga gaa cac agg ttc aga aac ttt tgc cgg 2782 Pro Leu Phe Ile Val Gly Arg Glu His Arg Phe Arg Asn Phe Cys Arg 840 845 850 gtg gtg gtc cga gca cgc ttc aac gca tct aaa aca gac cct gtc aca 2830 Val Val Val Arg Ala Arg Phe Asn Ala Ser Lys Thr Asp Pro Val Thr 855 860 865 gga gct gtg aaa aat aca aag tac cat caa ctt tat gat ttg ctg gga 2878 Gly Ala Val Lys Asn Thr Lys Tyr His Gln Leu Tyr Asp Leu Leu Gly 870 875 880 ttg gtc act tac ctg gac tgg gtc atg atc atc gta acc atc tgc tct 2926 Leu Val Thr Tyr Leu Asp Trp Val Met Ile Ile Val Thr Ile Cys Ser 885 890 895 tgc att tcc atg atg ttt gag tcc ccg ttt cga aga gtc atg cat gca 2974 Cys Ile Ser Met Met Phe Glu Ser Pro Phe Arg Arg Val Met His Ala 900 905 910 915 cct act ttg cag att gct gag tat gtg ttt gtg ata ttc atg agc att 3022 Pro Thr Leu Gln Ile Ala Glu Tyr Val Phe Val Ile Phe Met Ser Ile 920 925 930 gag ctt aat ctg aag att atg gca gat ggc tta ttt ttc act cca act 3070 Glu Leu Asn Leu Lys Ile Met Ala Asp Gly Leu Phe Phe Thr Pro Thr 935 940 945 gct gtc atc agg gac ttc ggt gga gta atg gac ata ttt ata tat ctt 3118 Ala Val Ile Arg Asp Phe Gly Gly Val Met Asp Ile Phe Ile Tyr Leu 950 955 960 gtg agc ttg ata ttt ctt tgt tgg atg cct caa aat gta cct gct gaa 3166 Val Ser Leu Ile Phe Leu Cys Trp Met Pro Gln Asn Val Pro Ala Glu 965 970 975 tcg gga gct cag ctt cta atg gtc ctt cgg tgc ctg aga cct ctg cgc 3214 Ser Gly Ala Gln Leu Leu Met Val Leu Arg Cys Leu Arg Pro Leu Arg 980 985 990 995 ata ttc aaa ctg gtg ccc cag atg agg aaa gtt gtt cga gaa ctt ttc 3262 Ile Phe Lys Leu Val Pro Gln Met Arg Lys Val Val Arg Glu Leu Phe 1000 1005 1010 agc ggc ttc aag gaa att ttt ttg gtc tcc att ctt ttg ctg aca tta 3310 Ser Gly Phe Lys Glu Ile Phe Leu Val Ser Ile Leu Leu Leu Thr Leu 1015 1020 1025 atg ctc gtt ttt gca agc ttt gga gtt cag ctt ttt gct gga aaa ctg 3358 Met Leu Val Phe Ala Ser Phe Gly Val Gln Leu Phe Ala Gly Lys Leu 1030 1035 1040 gcc aag tgc aat gat ccc aac att att aga agg gaa gat tgc aat ggc 3406 Ala Lys Cys Asn Asp Pro Asn Ile Ile Arg Arg Glu Asp Cys Asn Gly 1045 1050 1055 ata ttc aga att aat gtc agt gtg tca aag aac tta aat tta aaa ttg 3454 Ile Phe Arg Ile Asn Val Ser Val Ser Lys Asn Leu Asn Leu Lys Leu 1060 1065 1070 1075 agg cct gga gag aaa aaa cct gga ttt tgg gtg ccc cgt gtt tgg gcg 3502 Arg Pro Gly Glu Lys Lys Pro Gly Phe Trp Val Pro Arg Val Trp Ala 1080 1085 1090 aat cct cgg aac ttt aat ttc gac aat gtg gga aac gct atg ctg gcg 3550 Asn Pro Arg Asn Phe Asn Phe Asp Asn Val Gly Asn Ala Met Leu Ala 1095 1100 1105 ttg ttt gaa gtt ctc tcc ttg aaa ggc tgg gtg gaa gtg aga gat gtt 3598 Leu Phe Glu Val Leu Ser Leu Lys Gly Trp Val Glu Val Arg Asp Val 1110 1115 1120 att att cat cgt gtg ggg ccg atc cat gga atc tat att cat gtt ttt 3646 Ile Ile His Arg Val Gly Pro Ile His Gly Ile Tyr Ile His Val Phe 1125 1130 1135 gta ttc ctg ggt tgc atg att gga ctg acc ctt ttt gtt gga gta gtt 3694 Val Phe Leu Gly Cys Met Ile Gly Leu Thr Leu Phe Val Gly Val Val 1140 1145 1150 1155 att gct aat ttc aat gaa aac aag ggg acg gct ttg ctg acc gtc gat 3742 Ile Ala Asn Phe Asn Glu Asn Lys Gly Thr Ala Leu Leu Thr Val Asp 1160 1165 1170 cag aga aga tgg gaa gac ctg aag agc cga ctg aag atc gca cag cct 3790 Gln Arg Arg Trp Glu Asp Leu Lys Ser Arg Leu Lys Ile Ala Gln Pro 1175 1180 1185 ctt cat ctc ccg cct cgc ccg gat aat gat ggt ttt aga gct aaa atg 3838 Leu His Leu Pro Pro Arg Pro Asp Asn Asp Gly Phe Arg Ala Lys Met 1190 1195 1200 tat gac ata acc cag cat cca ttt ttt aag agg aca atc gca tta ctc 3886 Tyr Asp Ile Thr Gln His Pro Phe Phe Lys Arg Thr Ile Ala Leu Leu 1205 1210 1215 gtc ctg gcc cag tcg gtg ttg ctc tct gtc aag tgg gac gtc gag gac 3934 Val Leu Ala Gln Ser Val Leu Leu Ser Val Lys Trp Asp Val Glu Asp 1220 1225 1230 1235 ccg gtg acc gta cct ttg gca aca atg tca gtt gtt ttc acc ttc atc 3982 Pro Val Thr Val Pro Leu Ala Thr Met Ser Val Val Phe Thr Phe Ile 1240 1245 1250 ttt gtt ctg gag gtt acc atg aag atc ata gca atg tcg cct gct ggc 4030 Phe Val Leu Glu Val Thr Met Lys Ile Ile Ala Met Ser Pro Ala Gly 1255 1260 1265 ttc tgg caa agc aga aga aac cga tac gat ctc ctg gtg acg tcg ctt 4078 Phe Trp Gln Ser Arg Arg Asn Arg Tyr Asp Leu Leu Val Thr Ser Leu 1270 1275 1280 ggc gtt gta tgg gtg gtg ctt cac ttt gcc ctc ctg aat gca tat act 4126 Gly Val Val Trp Val Val Leu His Phe Ala Leu Leu Asn Ala Tyr Thr 1285 1290 1295 tac atg atg ggc gct tgt gtg att gta ttt agg ttt ttc tcc atc tgt 4174 Tyr Met Met Gly Ala Cys Val Ile Val Phe Arg Phe Phe Ser Ile Cys 1300 1305 1310 1315 gga aaa cat gta acg cta aag atg ctc ctc ttg aca gtg gtc gtc agc 4222 Gly Lys His Val Thr Leu Lys Met Leu Leu Leu Thr Val Val Val Ser 1320 1325 1330 atg tac aag agc ttc ttt atc ata gta ggc atg ttt ctc ttg ctg ctg 4270 Met Tyr Lys Ser Phe Phe Ile Ile Val Gly Met Phe Leu Leu Leu Leu 1335 1340 1345 tgt tac gct ttt gct gga gtt gtt tta ttt ggt act gtg aaa tat ggg 4318 Cys Tyr Ala Phe Ala Gly Val Val Leu Phe Gly Thr Val Lys Tyr Gly 1350 1355 1360 gag aat att aac agg cat gca aat ttt tct tcg gct gga aaa gct att 4366 Glu Asn Ile Asn Arg His Ala Asn Phe Ser Ser Ala Gly Lys Ala Ile 1365 1370 1375 acc gta ctg ttc cga att gtc aca ggt gaa gac tgg aac aag att atg 4414 Thr Val Leu Phe Arg Ile Val Thr Gly Glu Asp Trp Asn Lys Ile Met 1380 1385 1390 1395 cat gac tgt atg gtt cag cct ccg ttt tgt act cca gat gaa ttt aca 4462 His Asp Cys Met Val Gln Pro Pro Phe Cys Thr Pro Asp Glu Phe Thr 1400 1405 1410 tac tgg gca aca gac tgt gga aat tat gct ggg gca ctt atg tat ttc 4510 Tyr Trp Ala Thr Asp Cys Gly Asn Tyr Ala Gly Ala Leu Met Tyr Phe 1415 1420 1425 tgt tca ttt tat gtc atc att gcc tac atc atg cta aat ctg ctt gta 4558 Cys Ser Phe Tyr Val Ile Ile Ala Tyr Ile Met Leu Asn Leu Leu Val 1430 1435 1440 gcc ata att gtg gag aat ttc tcc ttg ttt tat tcc act gag gag gac 4606 Ala Ile Ile Val Glu Asn Phe Ser Leu Phe Tyr Ser Thr Glu Glu Asp 1445 1450 1455 cag ctt tta agt tac aat gat ctt cgc cac ttt caa atc ata tgg aac 4654 Gln Leu Leu Ser Tyr Asn Asp Leu Arg His Phe Gln Ile Ile Trp Asn 1460 1465 1470 1475 atg gtg gat gat aaa aga gag ggg gtg atc ccc acg ttc cgc gtc aag 4702 Met Val Asp Asp Lys Arg Glu Gly Val Ile Pro Thr Phe Arg Val Lys 1480 1485 1490 ttc ctg ctg cgg cta ctg cgt ggg agg ctg gag gtg gac ctg gac aag 4750 Phe Leu Leu Arg Leu Leu Arg Gly Arg Leu Glu Val Asp Leu Asp Lys 1495 1500 1505 gac aag ctc ctg ttt aag cac atg tgc tac gaa atg gag agg ctc cac 4798 Asp Lys Leu Leu Phe Lys His Met Cys Tyr Glu Met Glu Arg Leu His 1510 1515 1520 aat ggc ggc gac gtc acc ttc cat gat gtc ctg agc atg ctt tca tac 4846 Asn Gly Gly Asp Val Thr Phe His Asp Val Leu Ser Met Leu Ser Tyr 1525 1530 1535 cgg tcc gtg gac atc cgg aag agc ttg cag ctg gag gaa ctc ctg gcg 4894 Arg Ser Val Asp Ile Arg Lys Ser Leu Gln Leu Glu Glu Leu Leu Ala 1540 1545 1550 1555 agg gag cag ctg gag tac acc ata gag gag gag gtg gcc aag cag acc 4942 Arg Glu Gln Leu Glu Tyr Thr Ile Glu Glu Glu Val Ala Lys Gln Thr 1560 1565 1570 atc cgc atg tgg ctc aag aag tgc ctg aag cgc atc aga gct aaa cag 4990 Ile Arg Met Trp Leu Lys Lys Cys Leu Lys Arg Ile Arg Ala Lys Gln 1575 1580 1585 cag cag tcg tgc agt atc atc cac agc ctg aga gag agt cag cag caa 5038 Gln Gln Ser Cys Ser Ile Ile His Ser Leu Arg Glu Ser Gln Gln Gln 1590 1595 1600 gag ctg agc cgg ttt ctg aac ccg ccc agc atc gag acc acc cag ccc 5086 Glu Leu Ser Arg Phe Leu Asn Pro Pro Ser Ile Glu Thr Thr Gln Pro 1605 1610 1615 agt gag gac acg aat gcc aac agt cag gac aac agc atg caa cct gag 5134 Ser Glu Asp Thr Asn Ala Asn Ser Gln Asp Asn Ser Met Gln Pro Glu 1620 1625 1630 1635 aca agc agc cag cag cag ctc ctg agc ccc acg ctg tcg gat aga gga 5182 Thr Ser Ser Gln Gln Gln Leu Leu Ser Pro Thr Leu Ser Asp Arg Gly 1640 1645 1650 gga agt cgg caa gat gca gcc gac gca ggg aaa ccc cag agg aaa ttt 5230 Gly Ser Arg Gln Asp Ala Ala Asp Ala Gly Lys Pro Gln Arg Lys Phe 1655 1660 1665 ggg cag tgg cgt ctg cca tca gcc cca aaa cca ata agc cat tca gtg 5278 Gly Gln Trp Arg Leu Pro Ser Ala Pro Lys Pro Ile Ser His Ser Val 1670 1675 1680 tcc tca gtc aac tta cgg tta gga gga agg aca acc atg aaa tct gtc 5326 Ser Ser Val Asn Leu Arg Leu Gly Gly Arg Thr Thr Met Lys Ser Val 1685 1690 1695 gtg tgc aaa atg aac ccc atg act gac gcg gct tcc tgc ggt tct gaa 5374 Val Cys Lys Met Asn Pro Met Thr Asp Ala Ala Ser Cys Gly Ser Glu 1700 1705 1710 1715 gtt aag aag tgg tgg acc cgg cag ctg act gtg gag agc gac gaa agt 5422 Val Lys Lys Trp Trp Thr Arg Gln Leu Thr Val Glu Ser Asp Glu Ser 1720 1725 1730 ggg gat gac ctt ctg gat att taggtggatg tcaatgtaga tgaatttcta 5473 Gly Asp Asp Leu Leu Asp Ile 1735 gggtggaaac cgttttctaa taatgtcctt gattgtccag tgagcaatct gtaattgact 5533 ataactgaat tccagcttgt cacaagatgt ttataaattg attttcatcc tgccacagaa 5593 aggcataagc tgcagtatga tgggttacta tcaatcattg ctcaaaaaaa tttttgtata 5653 atgacagtac tgataatatt agaaatgata ccgcaagcaa atgtatatca ttaaaaatgt 5713 catatattct gtctgcgtaa actaagtata attcatattg ctctaatagt attatcaccg 5773 ccccsmaaag agtgctaagc ccaaagtggc tgatatttag ggtacagggg ttatagcttt 5833 agttcacatc tttcccattt ccactagaaa ttttctcgag agaatttatt atttatgatt 5893 gatctgaaaa ggtcagcact gaacttatgc taaatgatag tagttttaca aactacagat 5953 tctgaatttt aaaaagtatc ttctttttct cgtgttatat ttttaaatat acacaagaca 6013 tttggtgacc agaacaagtt gatttctgtc ctcagttatg ttaatgaaac tgttgcctcc 6073 ttctaagaaa attgtgtgtg caagcaccag gcaaagaaat ggactcagga tgcttagcgg 6133 tttaaaacaa acctgtagat aaatcacttg agtgacatag ttgcgcaaag atgttaagtt 6193 tcttaagaaa ccttttaata actgagttta gcaaaaagaa taaaactata tagctcaatt 6253 tatttaaaaa aatctttttg catgtgtgat gttatcattg gcttcatttc ttacccaagg 6313 tatgtctgtt ttgccataaa tcagcagagt catttcattc tgggtgatcc tgacacacca 6373 ttgctatgtt agatttgaaa tgacatctct gttaaaagaa tcttctatgg aaataatggt 6433 gccctgcaaa atctycctct gaactcacag gttagggatc acacaactta cttaatcgtt 6493 ttttgttttt gtttttttyc cttatatgtc aatcggccca tgtcctccgg gaaaattaga 6553 aaagcaaaat gattacaaag tgctgttaga tttcttgtgc tgggccagcc aagtagaagt 6613 ggacttgact tggaccttta actattttat tacagattgg acatttgctg ttcagatgtt 6673 ttttaacaga gggattatct cagaatcctg tgacctccag gttgttttat aatctatttt 6733 tctctattta acattcctca gatagatagg caaataggac attccttctg tgtcacagaa 6793 gtatcgtggt agtggcagtc tacagtttat atgattcatt gtaactatga gataaagaac 6853 aaccagtcat gtggccaaaa ggattagatt tgattggatg ttcacttgga gtttactttt 6913 tgtacataca agataaaata aatattggat ttgtaaaat 6952 3 1738 PRT Homo sapiens 3 Met Leu Lys Arg Lys Gln Ser Ser Arg Val Glu Ala Gln Pro Val Thr 1 5 10 15 Asp Phe Gly Pro Asp Glu Ser Leu Ser Asp Asn Ala Asp Ile Leu Trp 20 25 30 Ile Asn Lys Pro Trp Val His Ser Leu Leu Arg Ile Cys Ala Ile Ile 35 40 45 Ser Val Ile Ser Val Cys Met Asn Thr Pro Met Thr Phe Glu His Tyr 50 55 60 Pro Pro Leu Gln Tyr Val Thr Phe Thr Leu Asp Thr Leu Leu Met Phe 65 70 75 80 Leu Tyr Thr Ala Glu Met Ile Ala Lys Met His Ile Arg Gly Ile Val 85 90 95 Lys Gly Asp Ser Ser Tyr Val Lys Asp Arg Trp Cys Val Phe Asp Gly 100 105 110 Phe Met Val Phe Cys Leu Trp Val Ser Leu Val Leu Gln Val Phe Glu 115 120 125 Ile Ala Asp Ile Val Asp Gln Met Ser Pro Trp Gly Met Leu Arg Ile 130 135 140 Pro Arg Pro Leu Ile Met Ile Arg Ala Phe Arg Ile Tyr Phe Arg Phe 145 150 155 160 Glu Leu Pro Arg Thr Arg Ile Thr Asn Ile Leu Lys Arg Ser Gly Glu 165 170 175 Gln Ile Trp Ser Val Ser Ile Phe Leu Leu Phe Phe Leu Leu Leu Tyr 180 185 190 Gly Ile Leu Gly Val Gln Met Phe Gly Thr Phe Thr Tyr His Cys Val 195 200 205 Val Asn Asp Thr Lys Pro Gly Asn Val Thr Trp Asn Ser Leu Ala Ile 210 215 220 Pro Asp Thr His Cys Ser Pro Glu Leu Glu Glu Gly Tyr Gln Cys Pro 225 230 235 240 Pro Gly Phe Lys Cys Met Asp Leu Glu Asp Leu Gly Leu Ser Arg Gln 245 250 255 Glu Leu Gly Tyr Ser Gly Phe Asn Glu Ile Gly Thr Ser Ile Phe Thr 260 265 270 Val Tyr Glu Ala Ala Ser Gln Glu Gly Trp Val Phe Leu Met Tyr Arg 275 280 285 Ala Ile Asp Ser Phe Pro Arg Trp Arg Ser Tyr Phe Tyr Phe Ile Thr 290 295 300 Leu Ile Phe Phe Leu Ala Trp Leu Val Lys Asn Val Phe Ile Ala Val 305 310 315 320 Ile Ile Glu Thr Phe Ala Glu Ile Arg Val Gln Phe Gln Gln Met Trp 325 330 335 Gly Ser Arg Ser Ser Thr Thr Ser Thr Ala Thr Thr Gln Met Phe His 340 345 350 Glu Asp Ala Ala Gly Gly Trp Gln Leu Val Ala Val Asp Val Asn Lys 355 360 365 Pro Gln Gly Arg Ala Pro Ala Cys Leu Gln Lys Met Met Arg Ser Ser 370 375 380 Val Phe His Met Phe Ile Leu Ser Met Val Thr Val Asp Val Ile Val 385 390 395 400 Ala Ala Ser Asn Tyr Tyr Lys Gly Glu Asn Phe Arg Arg Gln Tyr Asp 405 410 415 Glu Phe Tyr Leu Ala Glu Val Ala Phe Thr Val Leu Phe Asp Leu Glu 420 425 430 Ala Leu Leu Lys Ile Trp Cys Leu Gly Phe Thr Gly Tyr Ile Ser Ser 435 440 445 Ser Leu His Lys Phe Glu Leu Leu Leu Val Ile Gly Thr Thr Leu His 450 455 460 Val Tyr Pro Asp Leu Tyr His Ser Gln Phe Thr Tyr Phe Gln Val Leu 465 470 475 480 Arg Val Val Arg Leu Ile Lys Ile Ser Pro Ala Leu Glu Asp Phe Val 485 490 495 Tyr Lys Ile Phe Gly Pro Gly Lys Lys Leu Gly Ser Leu Val Val Phe 500 505 510 Thr Ala Ser Leu Leu Ile Val Met Ser Ala Ile Ser Leu Gln Met Phe 515 520 525 Cys Phe Val Glu Glu Leu Asp Arg Phe Thr Thr Phe Pro Arg Ala Phe 530 535 540 Met Ser Met Phe Gln Ile Leu Thr Gln Glu Gly Trp Val Asp Val Met 545 550 555 560 Asp Gln Thr Leu Asn Ala Val Gly His Met Trp Ala Pro Val Val Ala 565 570 575 Ile Tyr Phe Ile Leu Tyr His Leu Phe Ala Thr Leu Ile Leu Leu Ser 580 585 590 Leu Phe Val Ala Val Ile Leu Asp Asn Leu Glu Leu Asp Glu Asp Leu 595 600 605 Lys Lys Leu Lys Gln Leu Lys Gln Ser Glu Ala Asn Ala Asp Thr Lys 610 615 620 Glu Lys Leu Pro Leu Arg Leu Arg Ile Phe Glu Lys Phe Pro Asn Arg 625 630 635 640 Pro Gln Met Val Lys Ile Ser Lys Leu Pro Ser Asp Phe Thr Val Pro 645 650 655 Lys Ile Arg Glu Ser Phe Met Lys Gln Phe Ile Asp Arg Gln Gln Gln 660 665 670 Asp Thr Cys Cys Leu Leu Arg Ser Leu Pro Thr Thr Ser Ser Ser Ser 675 680 685 Cys Asp His Ser Lys Arg Ser Ala Ile Glu Asp Asn Lys Tyr Ile Asp 690 695 700 Gln Lys Leu Arg Lys Ser Val Phe Ser Ile Arg Ala Arg Asn Leu Leu 705 710 715 720 Glu Lys Glu Thr Ala Val Thr Lys Ile Leu Arg Ala Cys Thr Arg Gln 725 730 735 Arg Met Leu Ser Gly Ser Phe Glu Gly Gln Pro Ala Lys Glu Arg Ser 740 745 750 Ile Leu Ser Val Gln His His Ile Arg Gln Glu Arg Arg Ser Leu Arg 755 760 765 His Gly Ser Asn Ser Gln Arg Ile Ser Arg Gly Lys Ser Leu Glu Thr 770 775 780 Leu Thr Gln Asp His Ser Asn Thr Val Arg Tyr Arg Asn Ala Gln Arg 785 790 795 800 Glu Asp Ser Glu Ile Lys Met Ile Gln Glu Lys Lys Glu Gln Ala Glu 805 810 815 Met Lys Arg Lys Val Gln Glu Glu Glu Leu Arg Glu Asn His Pro Tyr 820 825 830 Phe Asp Lys Pro Leu Phe Ile Val Gly Arg Glu His Arg Phe Arg Asn 835 840 845 Phe Cys Arg Val Val Val Arg Ala Arg Phe Asn Ala Ser Lys Thr Asp 850 855 860 Pro Val Thr Gly Ala Val Lys Asn Thr Lys Tyr His Gln Leu Tyr Asp 865 870 875 880 Leu Leu Gly Leu Val Thr Tyr Leu Asp Trp Val Met Ile Ile Val Thr 885 890 895 Ile Cys Ser Cys Ile Ser Met Met Phe Glu Ser Pro Phe Arg Arg Val 900 905 910 Met His Ala Pro Thr Leu Gln Ile Ala Glu Tyr Val Phe Val Ile Phe 915 920 925 Met Ser Ile Glu Leu Asn Leu Lys Ile Met Ala Asp Gly Leu Phe Phe 930 935 940 Thr Pro Thr Ala Val Ile Arg Asp Phe Gly Gly Val Met Asp Ile Phe 945 950 955 960 Ile Tyr Leu Val Ser Leu Ile Phe Leu Cys Trp Met Pro Gln Asn Val 965 970 975 Pro Ala Glu Ser Gly Ala Gln Leu Leu Met Val Leu Arg Cys Leu Arg 980 985 990 Pro Leu Arg Ile Phe Lys Leu Val Pro Gln Met Arg Lys Val Val Arg 995 1000 1005 Glu Leu Phe Ser Gly Phe Lys Glu Ile Phe Leu Val Ser Ile Leu Leu 1010 1015 1020 Leu Thr Leu Met Leu Val Phe Ala Ser Phe Gly Val Gln Leu Phe Ala 1025 1030 1035 1040 Gly Lys Leu Ala Lys Cys Asn Asp Pro Asn Ile Ile Arg Arg Glu Asp 1045 1050 1055 Cys Asn Gly Ile Phe Arg Ile Asn Val Ser Val Ser Lys Asn Leu Asn 1060 1065 1070 Leu Lys Leu Arg Pro Gly Glu Lys Lys Pro Gly Phe Trp Val Pro Arg 1075 1080 1085 Val Trp Ala Asn Pro Arg Asn Phe Asn Phe Asp Asn Val Gly Asn Ala 1090 1095 1100 Met Leu Ala Leu Phe Glu Val Leu Ser Leu Lys Gly Trp Val Glu Val 1105 1110 1115 1120 Arg Asp Val Ile Ile His Arg Val Gly Pro Ile His Gly Ile Tyr Ile 1125 1130 1135 His Val Phe Val Phe Leu Gly Cys Met Ile Gly Leu Thr Leu Phe Val 1140 1145 1150 Gly Val Val Ile Ala Asn Phe Asn Glu Asn Lys Gly Thr Ala Leu Leu 1155 1160 1165 Thr Val Asp Gln Arg Arg Trp Glu Asp Leu Lys Ser Arg Leu Lys Ile 1170 1175 1180 Ala Gln Pro Leu His Leu Pro Pro Arg Pro Asp Asn Asp Gly Phe Arg 1185 1190 1195 1200 Ala Lys Met Tyr Asp Ile Thr Gln His Pro Phe Phe Lys Arg Thr Ile 1205 1210 1215 Ala Leu Leu Val Leu Ala Gln Ser Val Leu Leu Ser Val Lys Trp Asp 1220 1225 1230 Val Glu Asp Pro Val Thr Val Pro Leu Ala Thr Met Ser Val Val Phe 1235 1240 1245 Thr Phe Ile Phe Val Leu Glu Val Thr Met Lys Ile Ile Ala Met Ser 1250 1255 1260 Pro Ala Gly Phe Trp Gln Ser Arg Arg Asn Arg Tyr Asp Leu Leu Val 1265 1270 1275 1280 Thr Ser Leu Gly Val Val Trp Val Val Leu His Phe Ala Leu Leu Asn 1285 1290 1295 Ala Tyr Thr Tyr Met Met Gly Ala Cys Val Ile Val Phe Arg Phe Phe 1300 1305 1310 Ser Ile Cys Gly Lys His Val Thr Leu Lys Met Leu Leu Leu Thr Val 1315 1320 1325 Val Val Ser Met Tyr Lys Ser Phe Phe Ile Ile Val Gly Met Phe Leu 1330 1335 1340 Leu Leu Leu Cys Tyr Ala Phe Ala Gly Val Val Leu Phe Gly Thr Val 1345 1350 1355 1360 Lys Tyr Gly Glu Asn Ile Asn Arg His Ala Asn Phe Ser Ser Ala Gly 1365 1370 1375 Lys Ala Ile Thr Val Leu Phe Arg Ile Val Thr Gly Glu Asp Trp Asn 1380 1385 1390 Lys Ile Met His Asp Cys Met Val Gln Pro Pro Phe Cys Thr Pro Asp 1395 1400 1405 Glu Phe Thr Tyr Trp Ala Thr Asp Cys Gly Asn Tyr Ala Gly Ala Leu 1410 1415 1420 Met Tyr Phe Cys Ser Phe Tyr Val Ile Ile Ala Tyr Ile Met Leu Asn 1425 1430 1435 1440 Leu Leu Val Ala Ile Ile Val Glu Asn Phe Ser Leu Phe Tyr Ser Thr 1445 1450 1455 Glu Glu Asp Gln Leu Leu Ser Tyr Asn Asp Leu Arg His Phe Gln Ile 1460 1465 1470 Ile Trp Asn Met Val Asp Asp Lys Arg Glu Gly Val Ile Pro Thr Phe 1475 1480 1485 Arg Val Lys Phe Leu Leu Arg Leu Leu Arg Gly Arg Leu Glu Val Asp 1490 1495 1500 Leu Asp Lys Asp Lys Leu Leu Phe Lys His Met Cys Tyr Glu Met Glu 1505 1510 1515 1520 Arg Leu His Asn Gly Gly Asp Val Thr Phe His Asp Val Leu Ser Met 1525 1530 1535 Leu Ser Tyr Arg Ser Val Asp Ile Arg Lys Ser Leu Gln Leu Glu Glu 1540 1545 1550 Leu Leu Ala Arg Glu Gln Leu Glu Tyr Thr Ile Glu Glu Glu Val Ala 1555 1560 1565 Lys Gln Thr Ile Arg Met Trp Leu Lys Lys Cys Leu Lys Arg Ile Arg 1570 1575 1580 Ala Lys Gln Gln Gln Ser Cys Ser Ile Ile His Ser Leu Arg Glu Ser 1585 1590 1595 1600 Gln Gln Gln Glu Leu Ser Arg Phe Leu Asn Pro Pro Ser Ile Glu Thr 1605 1610 1615 Thr Gln Pro Ser Glu Asp Thr Asn Ala Asn Ser Gln Asp Asn Ser Met 1620 1625 1630 Gln Pro Glu Thr Ser Ser Gln Gln Gln Leu Leu Ser Pro Thr Leu Ser 1635 1640 1645 Asp Arg Gly Gly Ser Arg Gln Asp Ala Ala Asp Ala Gly Lys Pro Gln 1650 1655 1660 Arg Lys Phe Gly Gln Trp Arg Leu Pro Ser Ala Pro Lys Pro Ile Ser 1665 1670 1675 1680 His Ser Val Ser Ser Val Asn Leu Arg Leu Gly Gly Arg Thr Thr Met 1685 1690 1695 Lys Ser Val Val Cys Lys Met Asn Pro Met Thr Asp Ala Ala Ser Cys 1700 1705 1710 Gly Ser Glu Val Lys Lys Trp Trp Thr Arg Gln Leu Thr Val Glu Ser 1715 1720 1725 Asp Glu Ser Gly Asp Asp Leu Leu Asp Ile 1730 1735 

1. An isolated and purified polynueleotide which encodes human BION1 which has an amino acid sequence as shown in SEQ ID NO:1 or a polymorphic variant found in a schizophrenic, Bipolar Disorder, or Panic Disorder Syndrome patient.
 2. The isolated and purified polynucleotide of claim 1 wherein the polynucleotide is a cDNA.
 3. The isolated and purified polynucleotide of claim 1 wherein the polynucleotide is genomic DNA.
 4. The isolated and purified polynucleotide of claim 1 wherein the polynucleotide is in a YAC clone.
 5. The isolated and purified polynucleotide of claim 1 wherein the polynucleotide is in a BAC clone.
 6. The isolated and purified polynucleotide of claim 4 wherein the YAC clone further comprises marker D135779.
 7. The isolated and purified polynucleotide of claim 1 wherein the polynucleotide comprises the amino acid-coding sequence shown in SEQ ID NO:2.
 8. The isolated and purified polynucleotide of claim 1 comprising the sequence shown in SEQ ID NO:2.
 9. The isolated and purified polynucleotide of claim 1 wherein the polynucleotide is a 6.2 kb transcript as measured on Northern blots.
 10. The isolated and purified polynucleotide of claim 1 which comprises a polymorphism shown in Table
 2. 11. The isolated and purified polynucleotide of claim 1 which comprises a polymorphism shown in Table
 3. 12. A method of determining susceptibility to Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome, comprising: determining a nucleotide at a defined location in a BION1 gene of a human; comparing the nucleotide at the defined location of the human to that of an affected family member having a polymorphism at the nucleotide; identifying the human as susceptible to schizophrenia, bipolar disorder, or panic disorder if the determined nucleotide contains the polymorphism found in the affected family member.
 13. The method of claim 12 wherein the family member is schizophrenic.
 14. The method of claim 12 wherein the family member has Bipolar Disorder.
 15. The method of claim 12 wherein the family member has Panic Disorder Syndrome.
 16. The method of claim 12 wherein the polymorphism is identified in Table 2 or Table
 3. 17. A method of determining susceptibility to Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome comprising: determining a nucleotide at a defined location in a BION1 gene of a human; if the determined nucleotide is a polymorphism previously identified as associated with Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome, identifying the human as susceptible to Panic Disorder Syndrome, Bipolar Disorder, or Schizophrenia.
 18. An isolated and purified polynucleotide comprising at least 18 contiguous nucleotides of a human BION1 coding sequence, wherein said polynucleotide comprises at least one codon identified in Table 1, which codon is for an amino acid found in human BION1 proteins at the corresponding position.
 19. An isolated and purified human BION1 protein comprising an amino acid sequence as shown in SEQ ID NO:1 or a polymorphic variant found in Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome patients.
 20. An isolated and purified human BION1 polypeptide comprising at least 6 amino acids of a human BION1 protein, wherein said polypeptide comprises an amino acid sequence found in humans but not in rats as identified in Table
 1. 21. An isolated and purified polynucleotide of a human BION1 gene, wherein said polynucleotide comprises a polymorphic nucleotide identified in Table 2 or Table
 3. 22. A vector comprising the polynucleotide of claim
 1. 23. A host cell comprising the vector of claim
 22. 24. A method of producing human BION1 comprising: culturing the host cell of claim 23 under conditions for expression of BION1 from the vector; collecting BION1 protein from the cultured host cells or culture medium.
 25. A method of screening test substances for candidates useful in treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome comprising: contacting a human BION1 protein with a test substance; determining which test substances bind to the human BION1 protein; wherein a test substance is identified as a candidate drug useful for treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome if it binds to human BION1.
 26. The method of claim 25 wherein the human BION1 protein has a polymorphic amino acid identified in Table 2 or Table
 3. 